Many viruses enter the cell via endocytosis and utilize the cell's the endosomal network as a means to infiltrate the cell and replicate. For example, viral entry into cells may be mediated by a viral glycoprotein (GP), which attaches viral particles to the cell surface, delivers them to endosomes, and catalyzes fusion between viral and endosomal membranes. For example, Rab9 GTPase was shown to be required for replication of HIV-1, filoviruses (such as Ebola and Marburg), and measles virus. Murray et al. 2005 J. Virology 79:11742-11751. Silencing Rab9 expression dramatically inhibited HIV replication, as did silencing the host genes encoding TIP47, p40, and PIKfyve, which also facilitate late-endosome-to-trans-Golgi vesicular transport. Reducing Rab9 expression also inhibited the replication of the enveloped Ebola and Marburg filoviruses and that of measles virus, but not the non-enveloped reovirus. US 20070087008 (Hodge et al.) describes RAB9A, RAB11A, and modulators of those proteins as potentially useful for decreasing viral replication, especially HIV replication.
Additional studies indicate that the endo/lysosomal cholestrol transporter Niemann-Pick C1 (NPC1) acts as a post-endocytic intracellular receptor that is necessary for Ebola and Marburg virus penetration. Carette et al., 2011 Nature 477:340. Niemann-Pick C1 (NPC1) and the homotypic fusion and vacuole protein sorting (HOPS) complex were identified in a genome-wide haploid genetic screen as host factors for filovirus entry. The NPC1 locus was the single strongest hit, with 39 independent insertions. The HOPS complex was the next strongest hit. Additional genes whose products are involved in the biogenesis of endosomes (PIKfyve) and lysosomes (BLOC1S1, BLOC1S2), and in the targeting of luminal cargo to the endocytic pathway (GNPTAB) were also identified, but only NPC1 was validated in functional assays. For example, NPC1 function was required for infection by Ebola and Marburg viruses in human fibroblasts, NPC1 deficiency conferred resistance to viral infection in HAP1 and CHO cells, and NPC1 null mice were resistant to infection and pathogenesis of Ebola and Marburg viruses. WO 2012/103081 (Chandran et al.) describes methods for treating filovirus infection using an agent that inhibits, inter alia, NPC1 and the HOPS.
In yeast, fusion of the phagosome membrane to the lysosome membrane requires the HOPs complex and phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). Synthesis of PI(3,5)P2 is mediated by phosphatidylinositol-3-phosphate 5-kinase (PIKfyve). Phosphoinositides such as PI(3,5)P2 are important lipid regulators of membrane trafficking and cellular signaling. Using the inhibitor YM201636, Jefferies et al. showed that inhibiting PIKfyve and blocking cellular production of PI(3,5)P2 disrupts endomembrane transport and retroviral budding. Jefferies et al. EMBO rep. 2008 9:164-170.
Ebola is a filamentous, lipid enveloped, negative stranded RNA virus of the family Filoviridae. There are five known strains of Ebola, four of which infect humans. The 5 strains are Bundibugyo, Sudan, Tai Forest, Zaire, or Reston; Reston is not known to infect humans. Zaire or Ebola virus is responsible for the majority of outbreaks (Burd, 2015). Initially Ebola targets macrophages and dendritic cells (Missai and Sullivan 2014); however, it is subsequently able to penetrate all cell types excluding lymphocytes by binding to the plasma membrane surface proteins. Viral entry is mediated by glycoprotein (GP), which attaches viral particles to the cell surface, delivers viral particles to the endosomes; and catalyzes fusion between the viral particles and the endosomal membranes (Gray, 2014, Cook and Lee, 2013, Ansari, 2014, Carette, et al. 2012).
EVD is global public health emergency and the World Health Organization has declared the recent epidemic to be a Public Health Emergency of International Concern. The NIH classifies EVD as a Category A pathogen which indicates that it poses the highest risk to public safety and national security (Stahelin, 2014). The recent West African outbreak was first reported in March 2014 (McCoy, et al. 2014, Meyers et al. 20152015, Gatherer, 2014, Ansari, 2014) with infection rates increasing by 13% in a 6 month period. The recent epidemic is concentrated in areas of West Africa in countries such as Guinea, Liberia, Nigeria, Senegal, and Sierra Leone. The current outbreak has killed over 3,800 people (documented cases) making it the most lethal outbreak of Ebola to date. In the recent outbreak, mortality rates are approximately 50% to 70%; while mortality rates in the 20 recognized outbreaks of Ebola range from 25 to 90% (Meyers et al. 2015). Clinical manifestations, duration of illness, case fatality rates and the degree of transmission rates in this outbreak are similar to previous outbreaks. Due to global travel and the international aid workers who have been on the ground in West Africa, infected people have traveled outside of West Africa. Countries such as the United Kingdom, Canada, Spain and the United States have allowed infected patients to re-enter and receive treatment. On Sep. 30, 2014, the CDC confirmed the first case of travel associated EVD in the US. Subsequently, two health care workers who were exposed to EVD through this initial patient, tested positive for EVD; these two cases were the first non-travel related cases of EVD in the United States.
EVD is initially transmitted to man through contact with an infected animal. The virus is spread in humans from direct contact with broken skin or mucous membranes with blood and body fluids including urine, saliva, feces, vomit, breast milk and semen; needles and syringes that are contaminated with the virus; or infected animals. EVD is not spread through the air, water or food nor is it believed to be transmitted through mosquitoes or other insect vectors. Symptoms of EVD may appear 2 to 21 days after exposure with an average of 10 days. During the latent period of the disease the patient may not show symptoms and is considered noncontagious.
Initial clinical signs of Ebola consist of: Fever (>38.6° C. or 101.5° F.), severe headache, muscle pain, weakness, diarrhea, vomiting, abdominal pain, and unexplained hemorrhage.
Over the clinical course of Ebola, patients can develop fulminant septic shock and coagulopathy which can subsequently lead to death. EVD is often masked upon clinical presentation because the early signs and symptoms of EVD are similar to other common viral infections; therefore, patient histories are critical to understanding the potential risk of EVD. Several diagnostic tests are available for Ebola; including RT-PCR and ELISA tests (Burd, 2015; Bishop 2014); however, none of these tests are available in a non-hospital setting; therefore, making confirmation of the diagnosis difficult in the field. EVD care is generally limited to supportive care including administration of IV fluids, maintenance of oxygen status and blood pressure and treatment other underlying conditions or organ dysfunction or failure. Recovery from EVD is dependent on the supportive care and immune response of the patient.
Currently, there are no approved therapies for EVD. Two experimental treatments for Ebola are available on an emergency use basis in the US. They are ZMAPP (MAPP Pharmaceuticals) which is a biologic composed of three humanized monoclonal antibodies and TKM-Ebola, (Tekmira) a siRNA which interferes with the viral proteins L, VP24, and VP35 (Bishop 2014).). The efficacy of both of these experimental agents has yet to be established in controlled clinical trials. Current supplies of ZMAPP are exhausted and the drug product requires frozen shipment while TKM-Ebola is on partial clinical hold due to safety issues; therefore, there exists an urgent medical need for the development of new safe and effective treatments.
The present invention addresses the need for antiviral compositions and methods for the treatment of subjects infected with viruses and the prophylaxis of subjects who are at risk for viral infection, and particularly for human subjects infected with or at risk of infection with Ebola virus.